The bacterial PEP:sugar phosphotransferase system (PTS) couples the phosphorylation and translocation of specific sugars across the membrane. The activity of the first protein in this pathway, enzyme I (EI), is regulated by a monomer-dimer equilibrium where the Mg(II)-dependent autophosphorylation by PEP requires the dimer. Dimerization constants for dephospho- and phospho-EI of the E. coli PTS as well as for mutants in which Glu or Ala is substituted for the active-site His189 [EI(H189E) or EI(H189A), respectively] have been measured under a variety of conditions by sedimentation equilibrium at pH 7.5, 4 and 20 C. Concurrently, thermal unfolding of these forms of EI have been monitored by differential scanning calorimetry and by changes in the intrinsic tryptophanyl residue fluorescence. Phosphorylated EI and EI(H189E) have 10-fold increased dimerization constants [log K = 6.3, expressed per M monomer] compared to those of dephospho-EI and EI(H189A) at 20 C. Dimerization is strongly promoted by 1 mM PEP with 2 mM Mg(II) [log K ? 10 at 4 or 20 C], as demonstrated with EI(H189A) which cannot undergo autophosphorylation. Together, 1 mM PEP and 2 mM Mg(II) also markedly stabilize and couple the unfolding of C- and N-terminal domains of EI(H189A) - increasing the transition temperature (Tm) for unfolding the C-terminal domain by ca. 18 K and that for the N-terminal domain by ca. 9 K to Tm = 336 K, giving a dissociation constant of ca. 0.003 mM for the dissociation of PEP from the C-domain at 45 C. PEP alone also promotes the dimerization of EI(H189A), but only increases Tm ca. 5 K for C-terminal domain unfolding without affecting N-terminal domain unfolding, giving an estimated dissociation constant of 0.2 mM for PEP dissociation in the absence of Mg(II) at 45 C. In contrast, the dimerization constant of phospho-EI is the same in the absence and presence of 5 mM PEP and 2 mM Mg(II). Thus, the separation of substrate binding effects from those of phosphorylation by studies with wild-type EI and the inactive EI(H189A) has shown that intracellular concentrations of PEP and Mg(II) are important determinants of both the conformational stability and dimerization of dephospho-enzyme I. This is a continuing project for which Roman H Szczepanowski and Sergei B. Ruvinov (who left the laboratory in early 2000) made essential contributions by expressing and purifying full-length E. coli enzyme I (wild-type and active-site mutants with Ala or Glu substituted for His189) as well as many of the biophysical measurements reported here. Mariana Dimitrova has made many new measurements including those with the inactive EI(H189A) in the presence of PEP and/or Mg(II). Final analysis of all data has been completed and a manuscript recently has been submitted to the journal BIOCHEMISTRY with M. N. Dimitrova as first author.